Ciguatoxins – Features, origin,

and methods for detection

Gambierdiscus toxicus

O

OO

O

O

O

O

O

O

O

O OO

R2

R1

OH

H

H

H

H

HHHO

HHHH

HH

HH

H H H

H

HH H

HO

Ciguatoxin (CTX) R1= -CH(OH)-CH2OH; R2=OH

CTX 4B R1= -CH=CH2; R2=H

H

Ciguatera Fish Poisoning (Ciguatoxins, Maitotoxins?)

Poisoning Cases

+About 20-50 thousand people suffered from the

poisoning every years by consumption of tropical and

subtropical reef fish. The most serious seafood

poisoning in terms of numbers of patients.

Ciguatera Fish Poisoning (Ciguatoxins, Maitotoxins?)

The name ciguatera was given by Don Antonio Parra in Cuba in

1787 to intoxication following ingestion of the “cigua”, the Spanish

trivial name of a sea snail, Turbo pica, reputed to cause indigestion.

The term “cigua” was transferred to an intoxication caused by the

ingestion of coral reef fishes.

More than 500 fish species have been involved in CFP.

Definition of Ciguatera Fish Poisoning

Previous definition

Seafood poisoning consisting of various symptoms

caused by consumption of tropical and subtropical reef

fish.

Recommended definition

Food Poisoning caused by ciguatoxin (CTX) analogues.

by the Food Safety Commission of Japan and EFSA

Panel on Contaminants in the Food Chain

Pacific CTXs (Type 1)

+P-CTXs consists of 13 rings.

+The E-ring consists of 7 atoms.

+CTX1B is the most potent toxin.

+CTX4A ,B are produced by Gamebierdiscus.

Pacific CTXs (Type 2)

+The E-ring consists of 8 atoms.

+CTX3C is produced by Gamebierdiscus.

Caribbean CTXs

+C-CTXs consists of 14 rings.

+The E-ring consists of 8 atoms.

Structure of Pacific (P) and Caribbean (C) cituatoxins.

P-CTX3C

C-CTX1

CTXs are consisting of 13 to 14 rings. C-CTX1 is less polar than P-CTXs.

CTXs are lipophilic heat-stable molecules that remain toxic after cooking or exposure to mild

acidic or basic conditions.

P-CTX1B analogues

Indian (I) –CTX ?

Na+=145 mM

K+=5 mM

Na+=10 mM

K+=140 mM

inside

outside

cell membrane

Na+

K+

U

U

U

outside

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

Na+

Na channel

Ciguatoxins

Brevetoxins

Paralytic shellfish toxins

Tetrodotoxins

Receptor-5

Receptor-1

Voltage-gated

sodium channel

Working mechanism of Ciguatoxins

Opening of the voltage-gated sodium channel

Channel

close

Channel

open

• Gastrointestinal (e.g. vomiting, diarrhoea, nausea),

• Neurological (e.g. tingling, itching)

• Cardiovascular (e.g. hypotension, bradycardia)

• Reversal of temperature.

• Based on case reports on human intoxications it appears that a concentration of 0.01 μg P-CTX1B equivalents/kg fish is expected not to exert effects in sensitive individuals when consuming a single fish meal.

• 70 ng P-CTX1B may cause symptoms for human.

• In severe cases the symptoms may begin as soon as 30 minutes after ingestion of contaminated fish, while in milder cases they may be delayed for 24 to 48 hours.

• Fatalities is very rare but may occur due to cardiorespiratory failure.

Symptoms

Toxicity Equivalent Factors (TEFs)

Toxins Acute i.p. LD50 in mice

P-CTX1B 1

P-CTX2 0.3

P-CTX3 0.3

P-CTX3C 0.2

2,3-dihydroxy P-CTX3C 0.1

51-hydroxy P-CTX3C 1

P-CTX4A 0.1

P-CTX4B 0.05

C-CTX1 0.1

C-CTX2 0.3

Causative species

Gambierdiscus is a genus of marine dinoflagellates that produce ciguatoxins, a type

of toxin that causes the foodborne illness known as ciguatera.

They are usually epiphytic on macroalgae growing on coral reefs.

The marine dinoflagellate genus Gambierdiscus occurs globally in tropic and

subtropic regions.

Currently, G. toxicus, G. polynesiensis, G. excentricus and G. silvae are recognized

as being highly toxic, but ongoing and future research might identify more toxic

compounds in other species.

Gambierdiscus toxicus

Photo from Wikipedia

P-CTXs produced by G. toxicus

Yogi et al. Anal Chem. 2011, 83

CTX3C

49-epiCTX3C

CTX4A

CTX4B

P-CTX Type 1

P-CTX Type 2

P-CTXs in several fish in Okinawa in Japan

Yogi et al. Anal Chem. 2011, 83

CTX1B

52-epi-54-

deoxyCTX1B

54-deoxy

CTX1B

• CFP has occurred frequently in Okinawa Islands.

• Ciguatoxin-1B (CTX1B), 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B have been detected in several fish (large red mumea, snapper, coral cod etc) implicated in food poisoning cases.

• CTX3C was detected in one individual coral cod (Oplegnathuspunctatus) collected in Miyazaki in Kyusyu Island.

• CFP has occurred by consumption of toxic fish meat while high toxicities were found in head and viscera.

• No fatal cases by CFP had been reported.

• CTX had not been detected in Gambierdiuscus in Japan.

• Japan has a ban on domestic sales of barracuda fish (MHWL, 1953) and several other fish species that are associated with CFP.

Japanese experiences on CFP

Analytical methods for CTXs

• Bioassays (mouse, chiken, cat, brine shrimp etc.)

• Cytotoxicity assay

• Sodium channel binding assay for CTXs

• ELISA

• LC/MS/MS

• Currently, there are no regulatory limits for CTX in fish but a guidance

level of 0.01 ppb P-CTX1B equivalent (0.1ppb C-CTX1 equivalent)

has been issued by the United States Food and Drug Administration

(US FDA).

• These recommendations were based on a 10-fold reduction of the

lowest concentration of CTX in meal remnants found to cause human

illness.

Mouse Bioassay (MBA)

• The MBA based on the method described by Banner et al. (1960) is

presently the most widely used assay for the detection of CTX-group

toxins in fish. The assay is described by Yasumoto et al. (1971, 1984)

and it has been extensively used in the Pacific area (Juranovic and

Park, 1991).

• The main advantages of the MBA are (1) the provision of a measure

of total toxicity based on the biological response of the animal to the

toxin(s), (2) it does not require complex analytical equipment.

• The main disadvantages of the MBA are (1) no specific information is

provided on individual toxins, (2) it is not sensitive enough to detect

relevant levels of CTX-group toxins, (3) it cannot be automated, (4) it

requires specialised animal facilities and expertise.

Cytotoxicity assay

• Cytotoxicity assays for the detection of CTX-group toxins in fish

tissues are based on the capacity of the toxins to bind to sodium

channels, causing them to open at normal cell resting membrane

potentials.

• This results in an influx of sodium ions, cell depolarisation and the

appearance of spontaneous action potentials in excitable cells.

• The reported cell based assay for the CTX-group toxins takes

advantage of this phenomenon to produce an assay that is highly

sensitive to CTX-group toxins.

• The LOQ of this assay for CTX-group toxins is at pg/kg shellfish level.

Sodium channel binding assay

• The assay measures the inhibition of the binding of [3H]-brevetoxin-3

to sodium-channels in rat brain synaptosomes in the presence of

CTX-group toxins.

Na+=145 mM

K+=5 mM

Na+=10 mM

K+=140 mM

inside

outside

cell membrane

Na+

K+

U

U

U

outside

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

U

Na+

Na channel

Ciguatoxins

Brevetoxins

Paralytic shellfish toxins

Tetrodotoxins

Receptor-5

Receptor-1

Voltage-gated

sodium channel

Working mechanism of Ciguatoxins

Opening of the voltage-gated sodium channel

Channel

close

Channel

open

ELISA

Enzyme Linked Immuno-Sorbent Assay

Immunity to disease

Virus, toxins

etc. (antigens)

macrophage

T

Helper T cell

Transmission

B

B cell

Production of

antibody

Transmission

antibody

Detection of PSP toxins by ELISA

①二枚貝の抽出液を添加

②標識貝毒を添加

③洗浄後に発色試薬を添加

貝毒抗体を固層化したプレート

（+）

（-）

④発色率による判定

貝毒標識貝毒

標識貝毒

有毒貝

無毒貝

Toxic

sample

Non-

toxic

sample

Antibody plate

PSP

Labeled

PSP

Labeled

PSP

Antibody

Coloring

reagent

Coloring

reagent

Symptom: No information

Maitotoxin activates Ca2+ permeable, non-selective cation channels,

leading to an increase in levels of cytosolic Ca2+ ions.

Gambierdiscus toxicus

H

Me

H

Me

H

Me

H

Me

Me

O

HO

OH

OH

Me

HO

HO

OH

OH

OH

Me OSO3Na

Me

OH

MeH

H

OH

H HMe

OH

H H H H H HOH

H HO

H

O

OH H H

O

O

O

O

O

O

OH

NaO3SO OH OHH

HO

HMe H HOHO

H

H

OH

O

OH

H

O

H

OH

HH

HOH

HOH

H

Me

OHH

HO

H

H

H

Me

HHHH

H

Me

Me

Me

OH

OH

H

MeMeMeH

O

O

O

OO

O

O

O

O

H

H

H

H

H

Me

MeHO

OH

O

O O

OO

O

O

O

O

O

Maitotoxins: MW 3422Water-soluble

MTX standard (0.5 μg/mL)

11.0 min

m/z 1689.8

Retention time (min)

Extracted ion chromatogram of MTX standard obtained by qTOF/LC/MS (m/z 1000-4000)

LC Condition

Columm Mightysil RP-18 GP φ2.0×250mm （5μm）

Mobile Phase A：Water 50 mM HCOOH

B：95% MeCN 2 mM HCOONH4

gradient step time(minute） 95%B

1 0-10 20-100

2 10-20 100

Equilibrate 7 20

Flow rate 0.3 mL/min

Columm oven 30℃

Injection volume 5 μL

Strain Speacies LocationMTX concentration

(µg/mL)

MTX per cell

（pg/cell）

KW070922 G. scabrosus Kouchi ND ND

G1G G. scabrosus Okinawa ND ND

K070922-2 G. scabrosus Kouchi ND ND

M080828-3 G. scabrosus Kouchi ND ND

T080908-1 G. scabrosus Kouchi ND ND

G18G G. scabrosus Okinawa 0.07 0.02

G19G G. scabrosus Okinawa ND ND

U1G G. scabrosus Okinawa ND ND

WI29G G. scabrosus Wakayama ND ND

M080828-2 Gambierdiscus sp. Type2 Kouchi ND ND

ON2G Gambierdiscus sp. Type2 Okinawa ND ND

OI4G Gambierdiscus sp. Type2 Kouchi ND ND

OI6G Gambierdiscus sp. Type2 Kouchi ND ND

S0080911-1 G. australes Kouchi 11.17 11.39

G2G G. australes Okinawa ND ND

I080606-1 G. australes Okinawa ND ND

M080828-4 G. australes Kouchi ND ND

S4G G. australes Kouchi 19.85 17.19

U2G G. australes Okinawa 2.84 7.31

M14G G. australes Kouchi ND ND

WI11G Gambierdiscus sp. type 3 Wakayama ND ND

WI9G Gambierdiscus sp. type 3 Wakayama ND ND

Maitotoxin content of cultured Gambierdiscus sp.